Mechanisms responsible for biliary acute pancreatitis are poorly understood primarily because (a) they cannot be studied using material derived from humans and (b) experimental models used in the past to explore these issues are not ideal. We have developed a model of necrotizing pancreatitis that is elicited by retrograde intraductal infusion of a bile acid (TLCS) into the mouse pancreatic duct and we believe that it is the most clinically relevant model of biliary pancreatitis developed to date. We have shown that TLCS-induced biliary pancreatitis in mice is a receptor-mediated disease that is dependent upon the G protein- coupled bile acid receptor-1 (Gpbar1) which is expressed on the apical surface of acinar cells. Our long-term goal is the development of clinical interventions that can effectively prevent or treat gallstone pancreatitis and the overall objective of this application, which is the next step toward attainment of our long-term goal, is to identify the mechanisms responsible for the pancreatitis-associated events that occur within acinar cells during the early stages of our clinically relevant model of biliary pancreatitis. Our central hypotheses is: (a) that autophagy, digestive zymogen activation, and acinar cell injury/death occur sequentially in biliary pancreatitis; (b) that they play critical roles in the evolution of biliary pancreatitis; and (c) that they may be potentially useful therapeutic targets. While those events are known to occur in the other, perhaps less clinically relevant, models previously employed, their underlying mechanisms in those models are poorly understood and, we believe, possibly not similar to the mechanisms that are operative in clinical biliary pancreatitis. The rationale for our proposed research is that our model of Gpbar1-mediated biliary pancreatitis can be used to elucidate clinically relevant mechanisms responsible for those events and that, once the mechanisms responsible for those events in biliary pancreatitis are known, interventions designed to prevent or treat biliary pancreatitis can be rationally designed. We plan to employ our model of biliary pancreatitis to test our central hypothesis and to accomplish the objective of this application by pursuing the following 3 specific aims: (1) Identify the mechanisms by which vacuolar ATPases regulate zymogen activation in biliary pancreatitis; (2) Define the role played by autophagy in biliary pancreatitis; and (3) Identify and characterize the major modes of acinar cell death in biliary pancreatitis. Aim#1 will focus on the role of V-ATPases in mediating zymogen activation while Aim #2 and #3 will employ genetically modified mouse strains in which autophagy is prevented by acinar cell- specific deletion of Atg5 and necroptosis is prevented by deletion of RIP3. We believe that our proposed research is highly innovative because it represents a new and major departure from the status quo for such studies. It will have a highly significant and positive impact on the field by permitting a major advancement in our understanding that would not otherwise have been possible and it will be the first step in a continuum of research leading to interventions designed to specifically prevent and/or treat acute biliary pancreatitis.